Cell line

ABSTRACT

A Namalwa cell line which is free of squirrel monkey retrovirus is useful for alpha-interferon production. The cell line can be used for the expression of recombinant polypeptides. It can be employed for packaging viruses for use in gene therapy.

The present invention relates to Namalwa cell lines and their use.

Human alpha-interferons (α-IFNs) belong to a multigene family located ona 400 kb segment of human chromosome 9. The family comprises at least 13genes encoding α-IFN subtypes. Each α-IFN protein consists of 165 or 166amino acid residues with molecular weights of between 18 and 26 kD(depending on the degree of glycosylation). Although chemicallydistinct, the subtypes are closely related with regions of highlyconserved amino acid sequences.

One commercially available source of α-IFN is human lymphoblastoid alphanl IFN (α n1 IFN). Which is a natural α-IFN. It is produced bystimulating a Namalwa human lymphoblastoid cell line with Sendai virusto produce a mixture of at least 13 subtypes of α-IFN, which are thenpurified by chromatography (U.S. Pat. No. 4,216,203; EP-A-0000520;EP-A-0097353). The number and quantity of particular subtypes is keptwithin defined limits during the manufacturing process.

This technique for producing α-IFN was in fact developed in the early1970's. It was during that period that the cell-line now used to produceall currently commercially available human lyphoblastoid interferon wasgenerated from a Burkitt's lymphoma tumour. This tumour was removed froma single individual named Namalwa, thus generating the eponymouslytitled cell line (Strander H. et al, J. Clin. Microbiol. 1, 116-117,1975 and Christofinis G. J. et al, J. Gen. Virol. 52, 169-171, 1981).Namalwa cells are publicly available under ATCC Deposit No.00001432-CRL.

Namalwa cells have also been used for the preparation of recombinantproteins (Yanagi H. et al, Gene 76, 19-26, 1989 and Okamoto M. et al,Bio/Technology 550-553, 1990). Namalwa cells are attractive for theproduction of recombinant proteins of human origin. Correctpost-translational processing of the recombinant human proteins can beexpected. Further, industrial-scale production of Namalwa cell lines iswell-established because of their use for producing α-IFN.

The possibility that there may be contaminants associated with productsobtained from a Namalwa cell line has been extensively researched.Namalwa cells have been tested for contamination with bacteria, virusesand mycoplasma. Some level of Epstein-Barr virus (EBV) have beendetected. Infectious virus is not formed, though. Further, EBV earlyantigen cannot be detected even when cells are treated with chemicalssuch as bromodeoxyuridine which can induce EBV replication.

However, the presence of genome of the squirrel monkey retrovirus (SMRV,a type D retrovirus) has been detected (Middleton et al, Int. J. Cancer52, 451-454, 1992). SMRV was first isolated from a squirrel monkey lungculture and displays an extended host range in vitro including canine,human, chimpanzee, rhesus monkey and mink cells. The virus is ofparticular interest since it has been identified as a contaminant of anumber of cell lines. To date, two strains of the virus, SMRV andSMRV-H, have been identified on the basis of differences in restrictionendonuclease patterns and sequence variation.

It is possible to ensure that no viable SMRV particles survive theproduction protocols that are employed to obtain products using Namalwacell lines. However, it is clearly preferable that no SMRV genome shouldbe present in Namalwa cells. SMRV genomes were detected by Middleton etal (supra) in all eight of a random selection of eight Namalwacell-lines sampled from laboratories world-wide. Hence it is consideredlikely that all prior, publicly available, Namalwa cells contain theSMRV genome.

Conclusively proving that particular cells are lacking a viralcontaminant is clearly dependent upon the sensitivity of the particularassay system employed. Direct assay for the presence of viral DNA bytechniques such as the polymerase chain reaction (PCR) may allowsensitivities of detection of up to one viral genome per 100 to 1000cells, even up to one viral genome per 1000 cells. If however a newcell-line is cloned from a single cell, this will of necessity containeither one or more viral genome per cell or none. Consequently thepossibility of obtaining a false negative assay result will be muchlower for such a cloned cell-line than for genetically heterogeneouscells.

Prior to the present invention, it was considered that the presence ofSMRV was an inherent characteristic of a Namalwa cell line. As mentionedabove, it is considered likely that all prior, publically available,Namalwa cell lines contain the SMRV genome.

Unexpectedly, we have located a SMRV-free Namalwa cell line. We alsounexpectedly found that Namalwa cell lines could be cloned successfullyby using high density, rather than low density, static cultures. Theresulting cloned Namalwa cell lines were shown to be SMRV-free. Eachcloned cell line had been derived from a single cell, thus providing aguarantee that the entire cell population of the cloned cell line wasSMRV-free.

Accordingly, the present invention provides a Namalwa cell line which isSMRV-free. Typically, the cell line is capable of producing aheterogenous population of α-IFN sub-types.

The cell line is preferably a cloned cell line. In that event, the α-IFNsub-types are generally substantially the same as those produced byexisting Namalwa cell lines that are used for the production ofcommercially available lymphoblastoid interferon. A cloned cell-line ofthe present invention is a genetically uniform population of cellsderived from a single cell and is a clone notwithstanding the occurrenceof any random mutation during the generation of the clonal populationfrom its single parent cell.

Preferably the α-IFN sub-types generated by a cell line of the presentinvention contain at least α-IFN sub-types 2b, 7, 10, 17 and 21; morepreferably 1, 2b, 5, 7, 8, 10, 14, 17 and 21. The α-IFN sub-types areadvantageously produced in the relative quantitative ranges indicated inTable 1 below. These ranges define the limits within which the subtypesof therapeutically acceptable lymphoblastoid interferon preferably fall.It is most preferred that a cell line generates an α-IFN sub-typeprofile substantially equivalent to that produced by the Namalwa cellsused for the production of Wellferon, for example by the cells formingthe subject of ATCC Deposit No. 00001432-CRL. This deposit was made atATCC, Rockville, Md. 20852, USA on 7th Jul. 1978.

                  TABLE 1                                                         ______________________________________                                               Sub-type                                                                             % by weight                                                     ______________________________________                                                1     1-16                                                                   2b     19-37                                                                   5     4-13                                                                   7 + 17 11-20                                                                   8     2-14                                                                   10     8-21                                                                   14     2-13                                                                   21     8-20                                                            ______________________________________                                    

Two SMRV-negative cell lines in accordance with the present inventionhave been made the subject of Budapest Treaty deposits at the EuropeanCollection of Animal Cell Cultures, Porton Down, Salisbury SP4 0JG, GBon 8th Dec. 1994 under accession nos. ECACC 94120840 and ECACC 94120841.Those two deposited cell lines and cell lines derived therefrom areparticularly preferred embodiments of the invention. Progeny of thedeposited cell lines thus form an aspect of the invention.

Useful cell lines according to the invention are SMRV-free cell linescapable of producing a heterogeneous population of α-IFN sub-types,which sub-types are substantially the same as those produced by eitherof deposited cell lines nos. ECACC 94120840 and ECACC 94120841. Thepopulation of sub-types, the IFN sub-type profile, may be substantiallythe same as that produced by either of these two cell lines. Therelative proportions of each α-IFN sub-type may thus be substantiallythe same as the relative proportions of each sub-type produced by eitherof the deposited cell lines.

We fortuitously located a SMRV-negative Namalwa cell line. The cell lineof the invention may thus be a naturally-occurring SMRV-free Namalwacell line. Such a cell line may be in isolated form. The cell line mayhave been cloned.

A cloned cell line of the invention can be prepared by a novel form ofdouble limiting dilution cloning. Contrary to known dilution cloningmethodologies, it was discovered that, when cells of suitable viabilitywere subsequently plated out in wells to obtain single colonies,advantageous results were obtained by using high density (rather thanthe expected low density) static cultures.

It is preferable to perform a double round of dilution cloning whengenerating a cloned cell line in order to obtain a very high statisticalprobability of up to 99.99% that the resultant cell line originates froma single cell. Parental SMRV-free Namalwa cells are grown in staticculture to high density in an appropriate medium containing a suitableantibiotic, split, diluted down and regrown to high density, discardingthose cultures not demonstrating>90% viability using a viability assaysuch as trypan blue exclusion. The cells are then split and diluted downagain.

The cell density should preferably be 1.8×10⁶ cells ml⁻¹ or more, forexample from 1.8×10⁶ to 2.4×10⁶ cells ml⁻¹ or from 2.0×10⁶ to 2.2×10⁶cells ml⁻¹. A suitable cell density is therefore approximately 2 millioncells ml⁻¹. Dilution down to 0.2 million cells ml⁻¹ is suitable.

However, a SMRV-negative Namalwa cell culture may be generated from aSMRV-positive Namalwa cell line by using recombinant DNA technology.Thus, methods such as directed homologous recombination can be used torecombine out the integrated SMRV genome and replace it with a piece ofnon-coding DNA.

Deletion of the SMRV genome from the relevant chromosome of aSMRV-positive Namalwa cell line is thus possible. The sequences flankingthe SMRV genome are identified. A vector is constructed which containsat least 3 kb of the flanking sequence to either side of the SMRVgenome. A targeting construct is made in which the region to be deletedis replaced by a positive selectable marker such as one encoding drugresistance, leaving the flanking DNA unaltered.

This construct, typically a plasmid, is then linearized and introducedinto the target SMRV-positive cells. Recombination occurs as a lowfrequency natural event, and cells in which this has been successful canbe selected by their ability to grow in the drug which should have beenintroduced by the targeting construct. Resistant clones must then beanalysed to ensure that drug resistance has not occurred by anon-specific mechanism, and that the SMRV genome has been successfullydeleted. It is possible to engineer the targeting construct in such away as to select against non-specific recombinants.

Once this has been completed one copy of the genome has been deleted. Ina heterozygous organism the second copy must also be removed. This isdone by the same procedure, with a targeting construct that may beidentical to the first one in all aspects, except that it contains adifferent drug resistant selectable marker. Double recombinants can thenbe selected that are resistant to both drugs, due to integration intoboth chromosomes.

Alternatively, it is possible that a sub-population of SMRV-free cellsmay exist in a SMRV-positive Namalwa cell line. A SMRV-positive Namalwacell line could thus be cloned. Clones of SMRV-free cells could then beidentified. The double limiting dilution cloning procedure above can beemployed.

The present invention also provides a process for producingalpha-interferon, which process comprises culturing a cell-line of thepresent invention and isolating the alpha-interferon thus produced. Aheterologous population of α-IFNs is thus obtained. Cell-lines of thepresent invention may be cultured according to any appropriate method.An interferon inducer such as Sendai virus may be added to the culture.The alpha-interferon may be isolated by affinity chromatography usinganti-IFN antibodies. A suitable polyclonal antibody for use in suchgeneration may be produced by techniques well known in the art (U.S.Pat. No. 4,216,203).

In more detail, a sample of a Namalwa cell line according to theinvention may be thawed from storage in liquid nitrogen and grown incultures of increasing size leading up to large scale culture. Asuitable culture medium may include RPMI 1640 supplemented with serum,such as gamma irradiated adult bovine serum. When the cell population isoptimal, sodium butyrate may be added to slow the growth rate of thecells and optimise the subsequent IFN yield (U.S. Pat. No. 4,216,203).The cells should then be induced to produce IFN, typically by theaddition of Sendai virus. The crude α-IFN is isolated and then passedthrough a purification procedure involving precipitations, solventextraction and chromatography. The final form has a purity of at least95% and a specific activity of approximately 100 IU/mg protein andconsists of at least 13 different subtypes of α-IFN (Finter N. B. et al,CSHSQB 101, 571-575, 1986).

Pharmaceutical formulations of the present invention comprise α-IFNproduced from cell lines of the present invention in admixture with apharmaceutically acceptable carrier. Preferably the carrier comprises amixture of sodium chloride, tris (Trimethamine US), glycine and humanalbumin solution. More preferably the sodium chloride is present at 8.5mg ml⁻¹, the Tris at 1.21 mg ml⁻¹, the glycine at 0.75 mg ml⁻¹ and thehuman serum albumin at a concentration resulting in a final proteinconcentration in the formulation of 1.5 mg ml⁻¹.

α-IFNs produced from cell lines of the present invention may be used inthe therapy of any known lymphoblastoid IFN-responsive condition and maybe administered by routes and in quantities well known in the art andsubstantially identical to those known for existing commercialtherapeutic lymphoblastoid IFNs.

For example in the treatment of hairy cell leukaemia, a dosing regime ofthree megaunits (3 MU) should be administered e.g. intravenously (iv)daily for between 12 and 16 weeks followed by 3 MU three times per weekuntil no hairy cells are detectable in the bone marrow. In hepatitis Btherapy, 10-15 MU may be administered three times weekly for between 4and 6 months while, for hepatitis C, 3 MU may be administered for up to6 months. These dosing regimes are accepted in the art but do notpreclude the use of alternative or higher dosing regimes should theparticular clinical factors dictate it.

A cell line of the invention can also be used as an expression systemwithin which recombinant proteins may be produced. Accordingly, thepresent invention provides:

a cell line according to the invention which harbours an exogenousexpressible DNA sequence encoding a polypeptide of interest;

a process for the preparation of such a cell line, which processcomprises transfecting a cell line according to the invention with anexpressible DNA sequence encoding the polypeptide of interest; and

a process for the preparation of a polypeptide of interest, whichprocess comprises maintaining a cell line harbouring an exogenousexpressible DNA sequence encoding the polypeptide of interest under suchconditions that the polypeptide is expressed and recovering theexpressed polypeptide.

A cell line capable of expressing a polypeptide of interest can beprepared utilising an expression vector which comprises a DNA sequenceencoding the polypeptide. The polypeptide is typically a humanpolypeptide, for example a growth factor. The polypeptide may beinsulin, erythropoietin, human growth hormone, GCSF, GMCSF, tissueplasminogen activator, urokinase, blood factor VIII, protein C, etc.

The expression vector is capable of expressing the polypeptide ofinterest when provided in SMRV-free cells according to the invention.Appropriate transcriptional and translational control elements areprovided, including a promoter operably linked to the DNA sequenceencoding the desired polypeptide. The promoter may be the SV40 (simianvirus 40) early promoter. An enhancer, a transcriptional terminationsite and translational stop and start codons are provided. A suitablesplice junction and a polyadenylation site may be present. The DNAsequence is provided in the correct frame such as to enable expressionof the polypeptide to occur.

The expression vector is thus a vector which is compatible with theSMRV-free Namalwa cells. If desired, a selectable marker gene ispresent. The expression vector is typically a plasmid. It generallycomprises an origin of replication.

SMRV-free Namalwa cells are transfected with the expression vector. Thetransfected cells are then maintained under conditions which enableexpression of the desired polypeptide to occur. The transfected cellsare cultured in an appropriate medium for this purpose. The desiredpolypeptide that has been expressed is isolated. The polypeptide ispurified as required.

The SMRV-free cell lines of the invention can further be used as apackaging/growth complementing cell line for viruses, for exampleretroviruses or adenoviruses. Accordingly, the present inventionprovides:

a cell line according to the invention which harbours sufficientexpressible viral genes to enable a corresponding viral vectorcomprising replication signals, a packaging sequence and a gene ofinterest to be packaged therein;

a process for the preparation of such a cell line, which processcomprises transfecting a cell line according to the invention withsufficient expressible viral genes to enable a corresponding viralvector comprising replication signals, a packaging sequence and a geneof interest to be packaged therein; and

a process for packaging viruses, which process comprises transfecting acell line which harbours sufficient expressible viral genes to enable acorresponding viral vector comprising replication signals, a packagingsequence and a gene of interest to be packaged therein with the saidviral vector and recovering the resulting packaged virus.

The packaged viruses can be used in gene therapy. These viruses areunable to replicate autonomously within a target cell but can deliver adesired gene to that cell.

The desired gene is initially provided in a viral vector whichadditionally comprises the viral replication signals and a packagingsequence, sometimes called the psi packaging sequence or anencapsidation sequence. The viral vector thus lacks, at least,functional sequences which are necessary for replication of the viruswithin an infected cell.

The cell line may be employed for packaging a retrovirus, in which casethe viral vector is a retroviral vector. Suitable retroviruses which maybe packaged include murine leukemia virus (MLV), gibbon ape leukemiavirus (GALV), reticuloendothelial virus (REV), spleen necrosis virus(SNV, mammalian C type), avian leukosis virus (ALV, avian C) and humanfoamy virus (HFV, spuma).

When the cell line is employed for packaging a retrovirus, the viralvector comprises long terminal repeat (LTR) sequences, the desired geneand a packaging sequence. Typically, therefore, the viral vector in suchcircumstances lacks functional gag, pol and env genes. These genes maybe missing entirely or in part or be present but renderednon-functional. The viral vector thus generally comprises a retroviralgenome lacking the appropriate functional genes but incorporating thegene of interest.

The desired gene is provided in the viral vector operably linked to apromoter. The gene may be a gene encoding a lymphokine or a cytokine. Itmay be a tumour suppressor gene, a corrective gene or a GDEPT (genedirected enzyme prodrug therapy) enzyme gene. The promoter may be acarcinoembryonic antigen (CEA), Muc-1, c-ErbB2, foliate binding protein(FBP) or vascular endothelial growth factor (VEGF) promoter. Terminationsequences may be provided as required.

The viral vector is introduced into a cell line according to theinvention which is provided with the viral functions that enablepackaging/growth complementation to occur. Thus, the cell line isprovided with the viral genes which are able to complement the functionsof the viral vector. These genes express the viral proteins that enablepackaging/growth complementation to occur, typically structuralproteins, replicative enzymes and regulatory factors.

When the cell line is used for packaging a retrovirus, typically, thegenes are the gag, pol and env genes. In particular, they are thefunctional viral genes that are missing from the viral vector.

The genes required by the cell line may be stably integrated into thecell genome. The genes may be provided in the form of proviral DNA. Anyappropriate technique may be employed to introduce the genes into thecell line.

In use, a cell line harbouring the viral genes required for packaging isinfected with a viral vector as above. The resulting packaged virus isthen collected from the cell culture. The collected virus may bepurified as desired prior to use in gene therapy.

The following Examples illustrate the invention. In the accompanyingdrawings:

FIG. 1 shows the reversed phase high pressure liquid chromatography(RPHPLC) profile determined for the uncloned SMRV-positive Namalwacell-line currently used in the production of Wellferon (RegisteredTrade Mark); and

FIG. 2 shows the profile determined for the cloned SMRV-free Namalwacell-line ECACC 94120841.

EXAMPLE 1 Testing of Namalwa Cell Line for the Presence of SMRV

1. General

A Namalwa cell line X which we located was tested for the presence ofthe SMRV genome. An assay procedure was adopted that used a genomic SMRVprobe that will detect SMRV at less than 1 genome copy per cell and willroutinely detect 1 genome copy per cell. Test DNA was cut withrestriction endonucleases that yielded diagnostic-sized fragments thatwere detected by DNA hybridisation followed by autoradiography.

In addition, a polymerase chain reaction (PCR) assay was done using twoSMRV/LTR specific oligonucleotide amplimers. Any amplified sequencewould be identified by hybridisation with an oligonucleotide probespecific for SMRV. Under the conditions used, the procedure will detectat least 1 genome copy per 2000 cells or better. As the PCR assay was infact negative, an additional assay employing nested primers within theSMRV genome was used. The nested primer set can detect SMRV to asensitivity of 1 genome copy per 10,000 cells or better.

2. Methods

(a) DNA Hybridisation

DNA Purification

A pellet consisting of approximately 7×10⁷ test cells was used for DNApreparation. The cell pellet was resuspended in proteinase-K lysisbuffer containing 20 μg ml⁻¹ RNase and 100 μg ml⁻¹ proteinase-K. Thesuspension was digested for 16 hours at 37° C. The deproteinised DNA wasextracted twice with phenol and twice with phenol-chloroform and finallyprecipitated by ethanol in the presence of ammonium acetate. The DNA wasrecovered by centrifugation at 3000 g for 30 minutes and the supernatantdiscarded. The pellet was washed in 70% ethanol and allowed to air dryfor 1 hour.

The DNA was allowed to redissolve in Tris EDTA (TE) buffer and thepurity and concentration of the DNA was assessed by spectrophotometry.The absorbance was measured at 280 nm and 260 nm. The 260/280 ratio was1.82 indicating that the sample DNA was pure. The yield of DNA wasestimated to be 315 μg.

Control DNAs

The negative control DNA consisted of purified human placental DNAcontaining no detectable SMRV sequences.

The SMRV positive control DNA consisted of human placental DNA withadded recombinant DNA, representing the complete SMRV genome, at 100,10, 1 and 0.1 genome equivalents. Genome equivalents (g.e.) werecalculated by assuming that the eukaryotic genome consists of 3×10⁹ bpand the SMRV genome is 8.4×10³ bp. In 10 μg of DNA one genome equivalentwould be 28 pg of SMRV DNA.

Further positive controls consisted of Namalwa DNA known to harbour SMRVand CCL 194 DNA, a mink lung cell containing SMRV sequences atapproximately 10 genome equivalents per cell.

The probe consisted of the full length SMRV genome isolated from theplasmid vector sequences.

(b) Preparation of DNAs for Hybridisation

10 μg samples of test DNA, negative control human placental DNA, SMRVpositive Namalwa DNA and CCL 194 DNA were digested to completion withthe endonuclease BamHI, BglII, EcoRI and PstI. Recombinant positivecontrols were constructed by adding 100, 0.10, 1 and 0.1 genomeequivalents of recombinant SMRV plasmid DNA to 10 μg human placentalDNA. These were then digested to completion with the endonuclease EcoRI.

The resulting DNA fragments were separated by electrophoresis through an0.8% agarose gel. The DNA was depurinated by a brief treatment with HCl,denatured in alkali and neutralized in situ before it was transferred toa charged nylon membrane by capillary blotting.

(c) Prehybridisation and Hybridisation

The membrane was prehybridised by incubating in hybridisation buffercontaining 50% formamide in 4×SSPE at 42° C. for 2 hours.

After prehybridisation the buffer was replaced with fresh hybridizationbuffer containing denatured SMRV DNA labelled to a high specificactivity with ³² P,

The hybridisation was conducted for 16 hours at 42° C. Followinghybridisation, the membrane was washed for 15 minutes at roomtemperature in 2×SSC/0.1% SDS, then for 15 minutes at 65° C. in2×SSC/0.1% SDS followed by 3×30 minute washes at 65° C. in 0.1×SSC/0.1%SDS. Autoradiography of the membrane was performed by exposing it toX-ray film for 24 hours and 72 hours.

(d) Oligonucleotide Primers and Probes

Oligonucleotide Amplimers and Probe

Position on the SMRV genome: Amplimers encompassing the tRNA bindingsite were used: amplimer 1 is from nucleotide 805-825; amplimer 2 isfrom nucleotide 1006-990 and the probe is internal to oligonucleotide863-888. The t-RNA binding site is from nucleotide 863-880.

Oligonucleotide Amplimers and Probe for Nested PCR

Position on the SMRV genome: for the outer primer set, amplimer 3 isfrom nucleotide 367-384 and amplimer 4 is from nucleotide 798-781. Theinner primer set consists of amplimer 1 from nucleotide 401-421 andamplimer 2 from nucleotide 604-588. The probe is internal to theamplimers from nucleotide 459-482.

Internal Control Amplimers

The internal control amplimers used were from the β-globin gene andwould amplify a DNA product of 205 bp.

(e) Sample Preparation

An aliquot of test cells were lysed by boiling in the presence of amatrix that efficiently absorbs lysis products that interfere with thePCR amplification process. The matrix was pelleted bymicrocentrifugation and the supernatant used for amplification. In allcases a positive displacement pipette was used which was designated foruse with negative controls and test DNA only.

(f) Preparation of PCR Reactions

Sentinel Controls

Triplicate sentinel controls consisting of the SMRV reaction mix with noDNA were prepared before the preparation of test DNA and negativecontrol samples. The tubes were left open for the duration of samplehandling to assess possible contamination.

Negative Control Samples and Test Article Samples

Negative control samples consisted of duplicate human placental DNAequivalent to 2×10⁵ cells (approximately 1 μg).

Test DNA samples consisted of duplicate partially purified DNAequivalent to 2×10⁵ cells (approximately 1 μg).

Positive Controls

Aliquots of human placental DNA were spiked with a dilution of the SMRVgenome equivalent to 1 copy in 500 cells (5.6 fg), 1 copy in 1000 cells(2.8 fg) and 1 copy in 2000 cells (1.4 fg). These aliquots were thenadded to the SMRV reaction mix. A further positive control consisting of1 μg of purified CCL194 DNA was also added to the SMRV reaction mix.

β-Globin Internal Control

Triplicate sentinel controls consisting of β-globin reaction mix wereprepared and aliquots of test DNA equivalent to 2×10⁵ cells were run inparallel with the SMRV reactions to ensure the DNA was able to beamplified.

(g) Preparation of Nested PCR Reactions

Primary (Outer) Reactions

Triplicate sentinel controls consisting of the primary (outer) SMRVreaction mix with no DNA were prepared before the preparation of testDNA and negative control samples. The tubes were left open for theduration of sample handling to assess possible contamination.

Negative control samples consisted of duplicate human placental DNAequivalent to 2×10⁵ cells (approximately 1 μg).

Test DNA samples consisted of duplicate partially purified DNAequivalent to 2×10⁵ cells (approximately 1 μg).

Aliquots of human placental DNA were spiked with a dilution of the SMRVgenome equivalent to 1 copy in 500 cells (5.6 fg), 1 copy in 1000 cells(2.7 fg) and 1 copy in 2000 cells (1.4 fg). These aliquots were thenadded to the reaction mix. A further positive control consisting of 1 μgof purified CCL 194 DNA was also added to primary (outer) SMRV reactionmix.

Secondary (Inner) Nested Reactions

After the first round of PCR (Primary) was finished, 2 μl (1/20) of eachreaction was removed and added to secondary (inner) SMRV reaction mix.

(h) PCR Reaction Conditions

The reaction conditions for PCR were as follows: β-globin and SMRV PCR:3cycles consisting of denaturation at 97° C. for 1 minute, annealing at55° C. for 45 seconds and extension at 68° C. for 1 minute. This wasfollowed by 30 cycles consisting of denaturation at 95° C. for 1 minute,annealing at 55° C. for 45 seconds and extension at 68° C. for 1 minute.A final extension of the DNA was done for 10 minutes at 68° C. aftercycling.

The reaction conditions for PCR with nested primers were as follows:

First Reaction

2 cycles consisting of denaturation at 95° C. for 3 min., annealing to55° C. for 45 seconds and extension at 72° C. for 2 minutes. This wasfollowed by 25 cycles consisting of denaturation at 95° C. for 1 minute,annealing at 55° C. for 45 seconds and extension at 68° C. for 1 minute.

Second Reaction

3 cycles consisting of denaturation at 97° C. for 1 minute, annealing at55° C. for 45 seconds and extension at 68° C. for 1 minute. This wasfollowed by 30 cycles consisting of denaturation at 95° C. for 1 minuteannealing at 55° C. for 45 seconds and extension at 68° C. for 1 minute.A final extension of the DNA was done for 10 minutes at 68° C. aftercycling.

(i) Electrophoresis and Hybridisation

Aliquots of the finished reactions were electrophoresed through a 5%(v/v) acrylamide gel (SMRV PCR products) or a 1.5% agarose gel (nestedPCR products), stained in ethidium bromide and examined and photographedunder UV light. The DNA was denatured, neutralised in situ andtransferred to charged nylon membranes by electroblotting (SMRV PCRproducts) or by capillary blotting (nested PCR products). The DNA wasbound to the membranes by baking at 80° C.

The membranes were pre-hybridised by incubation in hybridisation buffercontaining 5×SSC, and 7% SDS (w/v) at 50° C. for 2 h. Afterpre-hybridisation the buffer was replaced with hybridisation buffercontaining a ³² P-5'-end labelled oligonucleotide specific for SMRV. Thehybridisation was continued for 16 h at 50° C. Following hybridisationthe membranes were washed for 3×2 minutes in 5×SSC/0.1% SDS (w/v) atambient temperature followed by 3×30 minutes washes in 5×SSC/0.1 SDS(w/v) at 50° C. and a final wash of 5 minutes at 68° C. (Tm-4° C.).Autoradiography of the membrane was performed by exposing the filters toX-ray film for 16 h.

2. Validity and Results

(a) Validity

DNA Hybridisation

Under the hybridisation conditions used, the SMRV probe detected down toone genome equivalent of SMRV in a background of virus-negative humanplacental DNA. Sensitivity of detection for the virus would be of theorder of 0.1 g.e. per cell.

PCR

A valid test is defined as a test where appropriate amplification of thetarget sequences is detected in the positive control DNA and absent forall the negative controls. In addition, amplification of a genomictarget sequence must be seen in all samples. The test was valid:amplification of the target sequences was detected in the positivecontrol DNA and was absent in the negative controls. Amplification ofthe β-globin genomic target was detected in all the samples.

(b) Test Results: DNA Hybridisation

Absence of SMRV specific sequences in the test DNA: there was nohybridisation of the radiolabelled SMRV probe to the negative controlDNA or the test DNA under stringent conditions of hybridisation.

(c) Test Results PCR

β-Globin Internal Control

Analysis of the triplicate sentinel controls showed no visible amplifiedbands. However, each reaction mix containing DNA displayed a discreteband of the expected size of approximately 205 bp thereby indicatingthat the DNA was suitable for PCR amplification.

Negative Controls

No amplified DNA fragments could be seen in the three sentinel controlsor the negative control DNA reactions. No specific hybridisation of theradiolabelled probe to either the sentinel controls or the negativecontrol DNA was detected.

Positive Controls

The expected size of the amplified fragment from SMRV DNA would be 201bp (from nucleotide 805-1006). A discrete band of approximately 201 bpwas detected in the three recombinant positive control DNA lanescontaining 56 fg, 2.8 fg and 1.4 fg amounts of SMRV DNA and in the CCL194 positive control lane. This 201 bp was detected by the radiolabelledprobe indicating that the reactions had amplified the expectedfragments. The sensitivity of the assay is of the order of 1 g.e. in2000 cells.

Test DNA

No specific amplified DNA fragments were detected in the test DNAseither visually or following hybridisation with the radiolabelled probe.

(d) Test Results Nested PCR, Secondary (Internal) Reactions

Negative Controls

No amplified DNA fragments could be seen in the three sentinel controlsor the negative control DNA reactions. No specific hybridisation of theradiolabelled probe to either the sentinel controls or the negativecontrol DNA was detected.

Positive Control

The expected size of the amplified fragment from SMRV DNA would be 203bp (from nucleotide 401-604). A discrete band of approximately 203 bpwas detected in the three recombinant positive control DNA lanescontaining 5.6 fg, 2.8 fg and 1.4 fg amounts of SMRV DNA and in the CCL194 positive control lane. This 203 bp band was detected by theradiolabelled probe indicating that the reactions had amplified theexpected fragments. The band was detected in positive controlscontaining 1.4 fg, 0.7 fg, 0.35 fg, 0.175 fg and 0.087 fg dilutions ofDNA. These samples were not subjected to DNA hybridisation analysis. Thesensitivity of the assay is less than 1 g.e. in 10,000 cells.

Test DNA

No specific amplified DNA fragments were detected in the test DNAseither visually or following hybridisation with the radiolabelled probe.

3. Conclusion

The results are consistent with both the negative control humanplacental DNA and the test DNA from Namalwa cell line X being free ofSMRV.

EXAMPLE 2

SMRV negative Namalwa cells were cloned by double limiting dilution toachieve a 99.99% statistical probability that the derived clone wasgenerated from a single cell. Parental cells from Namalwa cell line Xwere grown in static culture in a medium comprising RPMI 1640 mediumsupplemented to 10% foetal bovine serum, 100 Uml⁻¹ penicillin, 100μgml⁻¹ streptomycin and 4 mM glutamine. The cells were grown in staticculture and split every 2-3 days; diluting in growth medium down to 0.2million cells ml⁻¹ and maintaining only cultures demonstrating>90%viability as indicated by trypan blue exclusion.

Only cells showing growth of 0.2 to 2 million cells ml⁻¹ in a three dayperiod were selected for dilution cloning and it was found to beessential that dilution cloning was carried out on cells which had grownto a density of about 2 million cells ml⁻¹. Selected cells were dilutedinto 96-well low-evaporation trays; plating out at between 1 and 300cells/well grown for three weeks, wrapped in aluminium foil.

Single colonies were picked from plates having less than 10% of wellswith single colonies and subjected to a second round of dilutioncloning. Double diluted clones were then compared by small scale IFNinduction using a cell concentration of 1 million cells ml⁻¹ in 12-welltrays, treating with 1 mM sodium butyrate and then adding 1 μl of Sendaivirus per well. IFN was harvested after a further 24 hours and assayedby ELISA. Those clones for which ELISA indicated a satisfactoryquantitative expression of IFN were further amplified in larger scalecultures/inductions and subjected to qualitative analysis of a α-IFNsubunit profile by reversed phase high pressure liquid chromatography(RPHPLC) to select those clones A, B, C, D, etc displaying the desiredprofile.

EXAMPLE 3

α-IFN produced and purified in accordance with standard manufacturingpractice (Lewis, W. G. and Finter, N. B. (1987), "The Production andPurity of a mixed human alpha interferon preparation (Wellferon) derivedfrom lymphoblastoid cells" in The Interferon System (Eds. Baron, S.,Dianzani, F., Stanton, G. J. and Fleischmann, W. R. Jnr). TexasUniversity Press) was analysed for RPHPLC generated subtype profile andas illustrated in FIG. 1.

EXAMPLE 4

Purification Protocol for Interferon Produced by Cloned Namalwa CellsDeposit Accession No. ECACC 94120841

A SMRV-negative clone was selected, ECACC 94120841, and was grown in3×300 ml shake flasks, and induced with Sendai virus after butyratetreatment according to standard protocols (Lewis, W. G. and Finter, N.B., supra). The culture supernatant was harvested and purified by twopasses on a purified anti-interferon antibody immunoaffinity column. Theinterferon was concentrated and analysed by RPHPLC to determine thesubtype composition which is illustrated in Table 2 and the profilewhich is shown in FIG. 2. It must be noted that the purity of theinterferon achieved in a small scale laboratory preparation was notcomparable to that of a production batch of interferon (Example 2 andFIG. 1) and therefore the values for peak are percent shown below may beinfluenced by the presence of non-interferon protein.

                  TABLE 2                                                         ______________________________________                                        Peak    α-IFN subtype                                                                       Peak area %                                                                              Acceptable range                               ______________________________________                                        1       14           5         2-13                                           2 + 3   2b          24         19-37                                          4       21          11         8-20                                           5        5           6         4-13                                           6       10          11         8-21                                           7 + 8   7 + 17      15         11-20                                          9        8          11         2-14                                           10 + 11  1           7         1-16                                           ______________________________________                                    

Table 2 shows the RPHPLC elution peak number with the correspondingα-IFN sub-type and the relative proportion ("Peak Area %") of eachsub-type obtained from the cloned cell-line together with, forcomparison, the range of each sub-type in therapeutically acceptablelymphoblastoid interferon.

By comparing FIGS. 1 and 2, a highly significant and sufficient level ofidentity can be seen between the IFN profiles for the SMRV-positiveNamalwa cell line currently used for the production of Wellferon(Registered Trade Mark) (FIG. 1) and the SMRV-negative Namalwa cell lineECACC 94120841. The interferon derived from the cell-lines can beconsidered to be substantially equivalent.

All references to α-IFN sub-types herein are consistent with thenomenclature of human interferon proteins approved by the InternationalSociety for Interferon and Cytokine Research and published in theJournal of Inteferon Research 14:223-226 (1994).

We claim:
 1. A Namalwa cell line which is free of squirrel monkeyretrovirus.
 2. A cell line according to claim 1 which produces apopulation of alpha-interferon sub-types comprising sub-types 2b, 7, 10,17 and
 21. 3. A cell line according to claim 2 wherein the sub-typescomprise sub-types 1, 2b, 5, 7, 8, 10, 14, 17 and
 21. 4. A cell lineaccording to claim 3, wherein the alpha-interferon sub-type 1 isproduced in an amount of 1-16% by weight, the alpha-interferon sub-type2b is produced in an amount of 19-37% by weight, the alpha-interferonsub-type 5 is produced in an amount of 4-13% by weight, thealpha-interferon sub-type 7+17 is produced in an amount of 11-20% byweight, the alpha-interferon sub-type 8 is produced in an amount of2-14% by weight, the alpha-interferon sub-type 10 is produced in anamount of 8-21% by weight, the alpha-interferon sub-type 14 is producedin an amount of 2-13% by weight, and the alpha-interferon sub-type 21 isproduced in an amount of 8-20% by weight.
 5. A cell line according toclaim 1 which produces a population of alpha-interferon sub-types whichare the same as those produced by cell line ECACC 94120840 or ECACC94120841.
 6. A cell line according to claim 5 wherein the population ofsub-types is substantially the same as that produced by cell line ECACC94120840 or ECACC
 94120841. 7. A cell line according to claim 1 which isfree of squirrel monkey retrovirus and is cell line ECACC 94120840 orECACC 94120841 or the progeny of either.
 8. A cell line according toclaim 1 which harbours an exogenous expressible DNA sequence encoding apolypeptide of interest.
 9. A cell line according to claim 1 whichharbours sufficient expressible viral genes to enable a correspondingviral vector comprising replication signals, a packaging sequence and agene of interest to be packaged therein.
 10. A process for thepreparation of alpha-interferon, which process comprises culturing acell line as defined in claim 1 and isolating the alpha-interferon thusproduced.
 11. A process for the preparation of a cell line as defined inclaim 8, which process comprises transfecting a cell line as defined inclaim 1 with an expressible DNA sequence encoding the polypeptide ofinterest.
 12. A process for the preparation of a polypeptide ofinterest, which process comprises maintaining a cell line as defined inclaim 8 under such conditions that the said polypeptide is expressed andrecovering the expressed polypeptide.
 13. A process for the preparationof a cell line as defined in claim 9, which process comprisestransfecting a Namalwa cell line which is free of squirrel monkeyretrovirus with sufficient expressible viral genes to enable acorresponding viral vector comprising replication signals, a packagingsequence and a gene of interest to be packaged therein.
 14. A processfor packaging viruses, which process comprises transfecting a cell lineas defined in claim 9 with a corresponding viral vector comprisingreplication signals, a packaging sequence and a gene of interest andrecovering the resulting packaged virus.